Fig. 1. Xenopus embryos at stage 35/36 (A) and stage 29/30 (B) or their u.v.-treated equivalent. From top to bottom;
normal embryo, IAD Type 1/2, IAD Type 3, IAD Type 4. -Lines en background grid are 1 mm apart.
Fig. 2. (A) Photomicrograph of an unstained transverse section of a normal stage 35/36 spinal cord. Note lateral marginal
zones and ventrally placed central canal. (B) Toluidine-blue-stained transverse section of a normal stage 35/36 IAD Type 1
spinal cord. Note central canal is now placed dorsally and the pale-staining marginal zone (*) lines the ventral half of the
cord. (C,D) Electron micrographs of the ventral midline of Type 1 spinal cord shown in B. Note the characteristic radial,
glia-like endfeet, normally only seen in the lateral marginal zones, the large extracellular spaces and, in D, several axonal
profiles lying immediately adjacent to the basal lamina (arrows). (E,F) Transverse sections of normal (E) and Type 1 spinal
cord (F) stained with the anti-vimentin antibody Z9. Vimentin-positive radial processes and endfeet are present only in the
lateral marginal zones of normal cords, but are present also across the ventral midline of the uv-treated cord. My,
myotomes; not, notochord; fp, floor plate; c, central canal; mz, marginal zone; ef, endfoot. Scale bar in A is 50 fan and
applies to A, B, E and F, and is 1um in C and D.
Fig. 3. Camera-lucida drawings of motoneurones. (A) Side-view whole-mounted spinal cord from a normal embryo.
Neurons filled from a myotome HRP application. Large black cell is a primary motor neurone and small black cell is a
secondary motor neurone. Positions and sizes of other motor neurone cell bodies are shown as unfilled outlines. Rohon-
Beard cells are shown stippled along the dorsal cord. Rostral is to the left. (B) Transverse sections from a normal embryo
showing two large primary motor neurones and three smaller secondary motor neurones. Note dendrites are restricted to
one side only. No motor neurones are found ventral to the neurocoel. (C,D,E) Ventral views of whole-mounted spinal
cords from u.v.-treated embryos. Motor neurone.s and their caudally running axons lie on or close to the ventral midline.
In C both cells have dendrites that clearly extend in both left and right halves of the marginal zone. Arrows indicate
positions where axons exit the cord. The most rostral cell in C is unusual in that the motor dendrites overlap with the
Rohon-Beard axons (shown dashed). One Rohon-Beard axon is shown crossing the cord ventrally; this was only seen once.
(F,G) Transverse sections from u.v.-treated embryos showing motor neurones with clear bilateral dendrites. Rohon-Beard
axons are shown as short wavy lines dorsolateral to the motor cells. Rohon-Beard and extramedullary cells are stippled.
(H,I) The position of motor neurones (ventrally) and Rohon-Beard and extramedullary cells (dorsally) plotted onto
standardised section outlines. H is from a normal embryo and I from a u.v.-treated embryo. Note the large number of cells
outside the spinal cord in I. My, myotome; not, notochord. Scale bar is 50um for B, C, H, I and 30 um for A, D, E, F, G.
Fig. 4. (A.B) Transverse sections of normal cords containing HRP-filled motor neurones. A shows two large, heavily filled
primary motor neurones and one small, pale secondary (arrowed). B shows a large primary and its dendrites confined to
the ipsilateral marginal zone. (C,D) Transverse sections of u.v.-treated cords containing large HRP-filled motor neurones
along the ventral midline. In C a labelled Rohon-Beard cell is present in the dorsolateral cord. (E,F) Transverse sections
of u.v.-treated cords containing HRP-labelled sensory and motor axons and several extrameduUary cells (curved arrows)
attached in lines or bunches to the dorsal cord surface. In E note that the sensory axons (filled straight arrows) occupy the
dorsolateralmost region of the ventral marginal zone and the motor axon (open arrow) lies midventrally. My, myotomes;
not, notochord; asterisk, melanocyte. Scale bar is 50j/m.
Fig. 5. Camera-lucida
drawings of neurones with
commissural axons. (A) Side
view, and (B) ventral view of
whole-mounted spinal cords
from normal embryos. Cells
labelled from HRP applied to
the contralateral rostral spinal
cord. A shows the two
morphological classes seen.
interneurones have multipolar
cell bodies and a thin ventrally
directed axon, while
(stippled) have unipolar cell
bodies and dendrites that arise
from a thicker ventrally
directed process. B shows that
the axons from these cells
cross the ventral surface at
angles close to 90° to the long
axis. (C) Side view and (D)
ventral view of whole-mounted
spinal cords from u.v.-treated
embryos. Cells labelled from
HRP applied to the
contralateral rostral cord. C
shows that both morphological
classes of commissural neurone
are present (compare with A).
D shows that the angle at
which their axon crosses the
ventral midline (shown dotted)
is more variable than in
control spinal cords. Scale bar
Fig. 6. Camera-lucida drawings of Kolmer-Agduhr cells. (A) Transverse section containing a Kolmer-Agduhr cell filled
caudal to a rostral cord HRP application in a normal embryo. This cell has a typically simple morphology, contacts the
cerebrospinal fluid and occupies a position adjacent or close to the floor plate cells. (B) Transverse section containing a
Kohner-Agduhr cell filled caudal to a rostral cord HRP application in a u.v.-treated embryo. This cerebrospinal fluid
contacting cell has characteristic simple morphology but now occupies a position close to the midline beneath the
neurocoel. (C) The positions of seven Kolmer-Agduhr cells from a u.v.-treated embryo have been superimposed on a
typical transverse section. All but one occupy a position close to the midline. (D) Side view of whole-mounted spinal cord.
In this u.v.-treated case, four Kohner-Agduhr cells have been filled rostral to a caudal HRP application. This is never seen
in normal embryos. The outline of the neurocoel is indicated, caudal is to the left and ventral at the bottom. Scale bar is
Fig. 7. Histograms to show the variability in motor
neurone size in control and u.v.-treated embryos. The large
numbers of small cells (less than 100 /xm2) are absent from
u.v.-treated embryos. Data collected from 5 control
animals and 4 experimental animals. The cross-sectional
area of cell bodies was measured from enlarged cameralucida
drawings of HRP stained motor neurones.
Fig. 8. Interneurones.
(A,B) Camera-lucida drawings
of transverse sections
containing neurones filled from
rostral cord (in B) and caudal
cord (in A) in u.v.-treated
embryos. Notice how many
cells have a clear dorsal to
ventral polarity of their main
process. Stippled cells are
Rohon-Beard cells and
extramedullary cells. Unfilled
cell profiles are from cells with
no clear processes and which
are thus probably oriented in a
(C) Photomicrograph of the
side view of a whole-mounted
u.v.-treated cord. HRP was
applied to the rostral cord (to
the left). Note the
orientation of most of the
labelled cells. Scale bar is
50 fim for A and B, and
100 um for C.
Fig. 9. Photomicrographs of the ventral surface of whole-mounted cords stained for glycinergic axons. A and B are from
control embryos, A from the rostral cord/caudal hindbrain area and B from midtrunk cord. Note the stained axons cross
the ventral cord in a fairly direct manner. C and D are from equivalent regions of a u.v. type 1 embryo. Note now how the
angle of crossing is highly variable. Scale bar is 50um and applies to all.
Fig. 10. Histograms to show the variability of angle of
crossing of glycinergic commissural axons in control and
u.v.-treated embryos. Angles were measured relative to the
ventral midline of the spinal cord, those directed rostrally
have values less than 90° and those directed caudally have
values greater than 90°. Angles were allocated into bin
widths of 10°. Note the increased variability in the
experimental cords and the increased proportion that are
directed caudally (hatched) rather than rostrally.
Fig. 11. Camera-lucida drawings of glycinergic cells and axons. (A,B) Ventral views of whole-mounted spinal cords from
u.v.-treated embryos. The positions of all glycinergic cell bodies within the drawn segments are indicated, but only some of
the axons are included for clarity. Most of the undrawn axons run longitudinally along the ventral surface of the cord and
would thus obscure other details. The arrowed axons have all grown dorsally and probably all arise from contralateral cell
bodies. This is clearly seen in three of the filled cells in A and in all four of the filled cells in B. The remaining two filled
cells in A have caudally directed axons which remain ipsilateral for unusually long distances before eventually crossing to
the other side of the cord. The growth cones of these cells were clear. Dotted lines represent axons growing on dorsal
surface of cord. (C,D) Side views of whole-mounted spinal cords from u.v.-treated embryos. Arrows indicate trajectories of
dorsally positioned glycinergic axons. In some cases (C) axons turn to grow back down ventrally on the same side of the
cord, while in others they continue circumferentially and grow over the dorsal surface and down ventrally on the
contralateral side of the cord (dotted in D). Stipple in C indicates position of large numbers of stained longitudinal axons.
Scale bar is 50 um.
Fig. 12. Motor neurone activity in paralysed embryos. (A) Control embryo. Extracellular action potentials recorded from
one left (L) and one right (R) intermyotome cleft region. Activity was evoked by a single electrical stimulus to the skin
(not shown). Note that the activity in left and right usually alternates. Occasionally short periods of synchronous activity
occurs, usually towards the start of an episode. Phase relations are indicated by dotted lines. (B) U.v.-treated embryo.
Extracellular potentials recorded from left and right intermyotome regions now show no periods of alternating motor
activity, rather the activity is always synchronous. Scale bar is 50um. syn, synchrony.